[125I]iodocyanopindolol binding), mRNA expression (assessed by Northern blotting), and gene transcription (using nuclear run-on assays) in rat lung. Dexamethasone (Dex) (0.2 mg/kg/d, days 1-8) increased beta 1- and beta2-receptor numbers by 70 and 69% above control, respectively, but did not change their mRNA expression. Isoproterenol (Iso) (0.96 mg/kg/d, days 2-8) decreased beta 1- and beta2-receptor numbers by 48 and 51%, respectively, and also reduced mRNA expression by 69 and 57%, respectively. The combination of Dex and Iso resulted in no net change in beta2-receptor number and its mRNA expression, although there was a significant reduction in beta 1-receptor number and mRNA expression. The mapping of beta 1- and beta2-receptors by receptor
Catecholamines play a central role in the regulation of energy expenditure, in part by stimulating lipid mobiliza- tion through lipolysis in fat cells. The beta-2 adrenoceptor (BAR-2) is a major lipolytic receptor in human fat cells. To determine whether known polymorphisms in codons 16, 27, and 164 of this receptor play a role in obesity and subcuta- neous adipocyte BAR-2 lipolytic function, we investigated a group of 140 women with a large variation in body fat mass. Only the polymorphisms in codons 16 and 27 were common in the study population. The Gln27Glu polymorphism was markedly associated with obesity with a relative risk for obesity of z 7 and an odds ratio of z 10. Homozygotes for Glu27 had an average fat mass excess of 20 kg and z 50% larger fat cells than controls. However, no significant associ- ation with changes in BAR-2 function was observed. The Arg16Gly polymorphism was associated with altered BAR-2 function with Gly16 carriers showing a fivefold increased agonist sensitivity and without any change in BAR-2 ex- pression. However, it was not significantly linked with obe- sity. These findings suggest that genetic variability in the human BAR-2 gene could be of major importance for obe- sity, energy expenditure, and lipolytic BAR-2 function in adipose tissue, at least in women. ( J. Clin. Invest. 1997. 100: 3005–3013.) Key words: fat cell • DNA • lipolysis • restriction fragment length polymorphism • polymerase chain reaction
Curvilinear fibrils with the tinctorial properties of amyloid were isolated from a patient with bone and joint involvement complicating chronic dialysis for renal disease. Subunit fractions of 24,000 and 12,000 mol wt were identified after gel filtration under dissociating conditions, the latter containing a significant amount of a dimer of the former. This was confirmed by Edman degradation of each fraction, which yielded the amino terminal sequence of normal human beta-2 microglobulin (B2M) to residues 20 and 30, respectively. The size of the subunit protein (12,000 mol wt) and the amino acid composition make it likely that intact B2M is a major constituent of the fibrils. B2M is thus another example of a low molecular weight serum protein, with a prominent beta-pleated sheet structure, that may adopt the fibrillar configuration of amyloid in certain pathologic states.
Our screening approach also enabled us to uncover a role for beta-2 adrenergic receptor signaling in modulat- ing DUX4 expression. This finding is unexpected based on our current understanding of D4Z4 regulation, and creates an important opportunity for future mechanistic studies to define the beta-2 adrenergic signaling path- ways involved and uncover additional targets for drug development. Our experiments so far suggest that the canonical PKA signaling pathway downstream of the beta-2 receptor may not be mediating the effects on DUX4. Though restraint must be taken when interpret- ing these data as residual PKA activity may be enough to mediate DUX4 suppression over the time course of our assay, it opens up the possibility that beta-2 agonists act on D4Z4 via alternative, PKA-independent pathways. It is interesting to speculate that these signaling cascades might impact chromatin modifiers that act on the D4Z4 array and therefore alter the epigenetic state of the locus (Fig. 7). Additionally, it is possible that alternative path- ways will be more specific for D4Z4, and therefore have fewer potential side effects if they can be identified and targeted for therapeutic modulation. Interestingly, several clinical trials of the beta-2 adrenergic agonist albuterol have already been carried out in FSHD patients based on the drug’s known anabolic effects [51–54]. Although albu- terol significantly increased muscle mass in these studies, this did not translate into functional improvements; however, better outcome measures to evaluate the effect- iveness of such treatments for FSHD are needed before any potential therapy is disregarded. Such measures are being developed and tested, and include better FSHD- relevant functional measurements, self-reported measures of disease burden, and non-invasive imaging of disease progression [55 – 57].
Hematopoietic stem cell interaction with elements of the underlying stroma is essential for sustained normal hematopoiesis. Here we have determined that adhesion receptors in the integrin family play a role in promoting adhesion of human hematopoietic stem cells to cultured human marrow stromal cells. Enriched CD34hi progenitor cells expressed VLA-4, VLA-5, and at least one or more beta2 integrins. Homogeneous marrow stromal cell
Int J DeL lliol 19 345 355 (1995) 345 Origil1al Article Inhibition of TGF B3 (but not TGF B1 or TGF B2) activity prevents normal mouse embryonic palate fusion CLARE L, BRUNET, PAUL M SHARPE and MARK W[.]
PLC-β2 upon stimulation by Rac. However, this mutant retained full stimulation by heterotrimeric G protein subunits. Therefore, targeted mutation of this key interfacial residue provides a means of dissecting the cellular function of Rac stimulated PLC-β2 signaling from that of G α q or G βγ . Knockdown of endogenous PLC- β 2 followed by transfection of this mutant into a hematopoeitic cell line where PLC- β 2 is exclusively expressed would therefore identify whether there is a role of this interaction in the processes of chemotaxis, phagocytosis, and superoxide production. Experiments aimed at utilizing this mutant to achieve such functional dissection are complicated by the expression of PLC- β 2 almost exclusively in hematopoeitic cells, which are notoriously difficult to transfect and manipulate. Previous attempts to do so in neutrophil-like (HL- 60) and macrophage-like (TP-1) cells have failed (data not shown).
Chronic pain is any pain that persists in the absence of normal pain-evoking stimuli. The pain can be intermittent or constant, and often does not have a clear cause. One of the main symptoms is hyperalgesia, heightened sensitivity to painful stimuli. However, sometimes sufferers experience allodynia, which is the sensation of pain to a normally non-painful stimulus. This heightened sensitivity to pain can impair an individual’s daily functioning by preventing activity, overwhelming the senses, or pervading thought and awareness. Chronic pain conditions are thought to affect as much as 25% of the US population at some point in their lives 117-119 . There is a clear psychological and physiological component 120 and there is evidence that genetic polymorphisms mediate pain sensitivity and risk for developing chronic pain conditions 66,121,122 . Clinically, the non-specific beta-blocker propranolol has been successfully used for treatment of migraine 123-128 fibromyalgia 129 , and rheumatoid arthritis 15,130 . Ultimately, development and maintenance of a chronic pain condition is influenced my many environmental, psychosocial, and genetic factors—making study of these conditions challenging.
Int I Dc\' lliol ~l 91 102 (1997) 91 O,igilla/ Artie/i' Differential effects of transforming growth factors B1, B2, B3 and B5 on chondrogenesis in mouse limb bud mesenchymal cells JESUS CHIMAL MONROY'[.]
Int J De\' BioI 35 17 24 (1991) 17 Original Ar/ide Immunodetection of the transforming growth factors B 1 and B2 in the developing murine palate AMY L GEHRIS, MARINA D'ANGELO, and ROBERT M GREENE' Dep[.]
Reverse transcription was performed in duplicate as described previously with oligo-dT primers , but using 1.5 µg total RNA and Superscript III reverse transcriptase (Invitrogen, Paisley, UK). Quantitative PCR for ERβ2 was performed on a Bio-Rad Icycler Real-Time PCR machine (Bio-Rad Laboratories Ltd., Hertfordshire, U.K.) using 4 µ1 of a 1/2 dilution of cDNA per reaction (equivalent to cDNA from approximately 150 ng of total RNA). ERβ2 PCR reactions included 1× IQ Supermix (Bio-Rad) and PCR primers and a Taqman probe (as given in Table 2). For control gene PCR (HPRT, GAPDH) and ERα 4 µl of a 1/50 dilution was used and the reaction contained IQ SYBR Green Supermix (Bio-Rad). The PCR reactions con- sisted of a hot-start Taq Polymerase activation step of 95°C for 3 minutes, followed by conditions shown to be produce unique, specific bands for each mRNA (Table 2). Expression levels of mRNA for each gene were calculated using standard curves produced with the relevant cloned cDNAs and correcting for the control genes (HPRT and GAPDH). All amplicons crossed introns to avoid amplifi- cation of genomic DNA and the identity of PCR products was confirmed by agarose gel electrophoresis and DNA sequence analysis, as described previously .
loci (37). Recent chromatin immunoprecipitation suggested that in addition to binding known sites in Eβ, the Runx1 protein also recognizes unidentified sites flanking both Dβ1 and D β 2 (38). Like the recently described D δ 2 promoter (39), the promoter region upstream of Dβ2 contains multiple potential E boxes, as well as consensus Runx binding sites at –948 and –219 (Fig. 4). Radiolabeled probes to both sites detected a complex pattern of binding activities when mixed with nuclear proteins from P5424 cells (Fig. 9, lane 1); a binding pattern markedly different than that seen when probes were mixed with proteins from mature B or T cell lines (lanes 9-10). Unlabeled Runx1-A or Runx1-B oligonucleotides were equally effective at inhibiting all of the binding complexes at both sites (lanes 2-3), whereas mutation of core ACC trinucleotide completely abolished each probe’s competitive ability (lanes 4-5). Expression of the three Runx proteins varies during early thymopoiesis. Whereas Runx1 peaks in DN3 cells, Runx2 and Runx3 expression declines throughout DN development (40). Consistent with the pattern of Runx expression, nucleoprotein complexes formed between Runx1-A or Runx1-B and proteins from the DN3-staged P5424 cells were exclusively supershifted by Runx1 Ab (lane 6), and not by antibodies to Runx3 (lane 7) or Runx2 (data not shown). Subsequent supershift analyses of the binding patterns at Runx1-A and –B with B and T cell protein extracts (lanes 9-10) suggested that both sites are occupied by Runx3 in mature B cells and by Runx2 in mature T cells (data not shown).
Beta 1- and beta2-adrenergic receptor (beta-ARs) expression in the thick ascending limb of rat kidney was studied at the level of mRNA and receptor coupling to adenylyl cyclase. Absolute quantitation of beta 1- and beta2-AR mRNAs in microdissected nephron segments was performed with an assay based on reverse transcription and polymerase chain reaction, using in vitro transcribed mutant RNAs as internal standards. In the cortical thick ascending limb (CTAL), the number of mRNA molecules/mm of tubular length was 2,806 +/- 328 (n = 12) for beta 1-AR and 159 +/- 26 for beta2-AR (P < 0.01). Lower levels were obtained in the medullary thick ascending, beta 1-AR mRNA still being predominant. The pharmacological properties of beta-ARS was also studied in the CTAL. Cyclic AMP accumulation was stimulated by beta-agonist with a rank order of potency of isoproterenol > norepinephrine > epinephrine. This observation, and the higher efficiency of a beta 1 than of a beta2 antagonist to inhibit isoproterenol-induced cAMP accumulation, establish the
epinephrine and the neuropeptide, bombesin, resulted in a potentiation of gastrin release. It was concluded that the stimulatory effect of the sympathetic system on gastrin release was mediated through beta2-adrenergic receptors. The data indicated that adrenaline released from the adrenal medulla and gastrin releasing peptide (the mammalian homolog of
Methods: In this investigation the validity of a wireless dry electrode system compared to a conventional wet electrode system was assessed, while addressing methodological limitations. In Experiment 1, the signal output of both EEG systems was examined at Fz, C3, Cz, C4, and Pz using a conductive head model and generated test signals at 2.5 Hz, 10 Hz, and 39 Hz. In Experiment 2, two-minutes of eyes-closed and eyes-open EEG data was recorded simultaneously with both devices from the adjacent electrode sites in a sample of healthy adults. Results: Between group effects and frequency*device and electrode*device interactions were assessed using a mixed ANOVA for the simulated and in vivo signal output, producing no significant effects . Bivariate correlation coefficients were calculated to assess the relationship between electrode pairs during the simultaneous in vivo recordings, indicating a significant positive relationship (all p 's < .05) and larger correlation coefficients ( r > ± 0.5) between the dry and wet electrode signal amplitude were observed for theta, alpha, beta 1, beta2, beta 3, and gamma in both the eyes-closed and eyes-open conditions.
Infiltrating T cells around salivary glands in the lips of Sjögren's syndrome (SjS) patients are crucial in the pathogenesis of this disease. To analyze the nature of infiltrating T cells, their T cell receptor repertoire was examined with quantitative polymerase chain reaction. The repertoire of V beta transcripts in lips of SjS was not restricted; however, the V beta2 and V beta 13 genes were predominantly expressed on the T cells of lip specimens in six and four of seven lips, respectively. Predominance of these genes was specific in lips because no predominant V beta transcripts were found in lips from healthy subjects and PBLs from SjS patients. These results indicated that the V beta2- and V beta 13-positive T cells expanded specifically and preferentially in SjS lips, thereby suggesting the possible role in triggering the autoimmunity of this disease.
The presence and properties of serum autoantibodies against beta-adrenergic receptors in patients with idiopathic dilated cardiomyopathy were studied using synthetic peptides derived from the predicted sequences of the human beta-adrenergic receptors. Peptides corresponding to the sequences of the second extracellular loop of the human beta 1- and beta2-adrenergic receptors were used as antigens in an enzyme immunoassay to screen sera from patients with dilated cardiomyopathy (n = 42), ischemic heart disease (n = 17), or healthy blood donors (n = 34). The sera of thirteen dilated cardiomyopathy patients, none of the ischemic heart disease patients, and four of the healthy controls monospecifically
Some remarks on the present results need to be addressed. First, there were no carbapenemase-producing strains in the panel, as expected considering that these organisms are still rare in France as in most European countries (23). However, the spread of carbapenemase-producing Enterobacteriaceae in some coun- tries would justify accurate and rapid tests to detect such strains in addition to 3GC resistant strains (24). Second, the evaluation of the test on P. mirabilis must be treated with caution, since no P. mirabilis strain classified as resistant to 3GC was included in the present study. However, 3GC resistance in this species is generally uncommon. Third, the test was evaluated in teaching hospitals with high prevalences of resistant strains. In situations of preva- lence of 3GC resistance below 10% (e.g., in some community set- tings), the PPV could be somewhat lower, e.g., 92% for 5% prev- alence of resistance. Last, the study found 2.4% noninterpretable results, mainly in species naturally producing inducible AmpC beta-lactamases (e.g., Enterobacter spp.) (Table 2). Noninterpre- table results could limit the interest of BLT for use in situations of higher prevalence of such species.
wavelength, is necessary for down-converter materials for hybrid LED applications. 28–30 In order to determine whether the synthesised linear oligouorene-BODIPYs were eﬀective in this role, photoluminescence measurements were recorded in dilute dichloromethane solutions by excitation at the wavelength corresponding to the maximum absorption for the oligo- uorene fragment for each compound respectively. The results are shown in Fig. 3 and summarised in Table 1. Our hypothesis that eﬃcient energy transfer from the oligouorene chain to the BODIPY unit would occur was proven to be correct, with each compound exhibiting uorescence from the BODIPY unit whilst under excitation of the oligouorene chain. Once more, comparison within each substitution series (either meso- or beta-) is useful. For example, the meso-substituted compounds, meso-TFBOD and meso-QFBOD, exhibited an intense, narrow emission band at 537 nm. In contrast, the beta-substituted series exhibited a broad red-shied emission from the BODIPY fragments at 563–564 nm, with a Stokes shi of 50–51 nm vs. 10–11 nm for the meso-substituted analogues. The aforemen- tioned featureless long wavelength absorption bands of the beta-substituted compounds, as well as the increased Stokes shis, are evidence of a signicant diﬀerence in the structure of the ground and the rst excited state aer structural relaxation. Recently, beta-substitution of BODIPY has been used as an approach for creating materials with a large Stokes shi due to geometry relaxation of the excited state. 34 However, in the case